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Purification, Cloning and Expression of Spinach Leaf Sucrose-Phosphate Synthase in Escherichia Coli

Purification, Cloning and Expression of Spinach Leaf Sucrose-Phosphate Synthase in Escherichia Coli

U Sonnewald, W P Quick, E MacRae, K P Krause, M Stitt

Planta. 1993 Feb;189(2):174-81.

PMID: 7763376

Abstract:

Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylene glycol and chromatography over diethylaminoethylcellulose, omega-aminohexyl-agarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat.(mg protein)-1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121-1130). Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP58856738	ω-Aminohexyl-Agarose		58856-73-8	Price

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